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HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions.

ABSTRACT: The vitamin D receptor/retinoid X receptor-? heterodimer (VDRRXR?) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1?,25-dihydroxyvitamin D3 (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-? heterodimer (VDRRXR?) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXR? and give mechanistic insights into how VDRRXR? controls the transcriptional activity of specific genes.

SUBMITTER: Zheng J

PROVIDER: S-EPMC5640644 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions.

Zheng Jie J Chang Mi Ra MR Stites Ryan E RE Wang Yong Y Bruning John B JB Pascal Bruce D BD Novick Scott J SJ Garcia-Ordonez Ruben D RD Stayrook Keith R KR Chalmers Michael J MJ Dodge Jeffrey A JA Griffin Patrick R PR

Nature communications 20171013 1

The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin D<sub>3</sub> (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present st ...[more]

PMID: 29030554

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Project description:Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded ?-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent ?-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.

Dataset Information

HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions.

Publications

HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions.

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